The purpose of this application is to investigate, at the molecular and integrative levels, the biology of the newly cloned mu and delta opioid receptors, attempting to understand how these molecules function as units, and how they interact with other brain molecules to modulate critical functions such as pain control and drug reward. A major emphasis is to understand the function of these receptors in the context of the multiplicity of ligands in the endogenous opioid family. Given that mu and delta receptors interact, to varying extents, with the products of the three opioid precursor gene family, and given that the endogenous ligands share a common "message" sequence, the question arises as to how the receptors achieve their affinity and selectivity for the various ligands. Thus, the first specific aim of this application is to carry out structure-function studies of the mu and delta receptors, investigating the nature of the binding pocket for the peptides and alkaloids, and the mechanisms of discrimination or selectivity among the various type of agonist ligands, and between agonists and antagonists. These studies will be conducted using molecular modeling and site-directed mutagenesis, as well as relying on an empirical approach with construction of chimeras to indicate critical domains, followed by mutagenesis of unique residues within these domains. The second specific aim is to place these opioid receptors in their anatomical context and to describe their expression relative to the expression of the 3 opioid precursors. Using dual in situ hybridization, receptor immunohistochemistry, or a combination of in situ and immunohistochemistry, we will study the expression of the mu and delta receptors and their ligands in order to determine the existence of autoreceptors, and to describe the interface between the opioid transmitters and the two receptors. We will survey the brain and spinal cord, and then focus on the anatomy of the opioid system in areas implicated in nociception and analgesia. This information will form the foundation of regulatory studies whereby stimuli known to alter pain responsiveness will be used to modulate the expression of the opioids, the receptors, or both. The third specific aim will move our knowledge of the mu and delta receptors at the molecular and anatomical level to a more complete description of the anatomy of drug reward, the role of opioid receptors in the reward circuits, and the modulation of these molecules by treatment with drugs such as morphine and cocaine. Using a combination of anatomical tools, we shall describe the co-expression of opioid molecules with other molecules relevant to reward mechanisms, particularly dopamine receptors. We shall define, at a finer level, subpopulations of cells in the nucleus accumbens with unique combinations of these molecules, and will study their projections. Then we shall relate these anatomical/biochemical entities to drug responsiveness, and determine changes in expression under conditions which promote drug tolerance versus drug sensitization. Taken together, these studies will permit us to use the cloning of the opioid receptors to achieve a better understanding of the biology of opioids and their role in drug use and abuse.